The overall hypothesis is: thyroglobulin (Tg) and its processing vary among normal subjects and in association with physiological and pathological stimuli. Thyroglobulin is the protein matrix for thyroid hormone synthesis and the vehicle for subsequent hormone storage. Further processing of Tg by enzymatic digestion is necessary to release hormone into the circulation. Previous work has suggested variations in Tg structure both among normals and in thyroid diseases, but the characterization of these changes has been limited. Thus the long-range objectives of this proposal are to define how Tg participates in the synthesis and intrathyroidal metabolism of thyroid hormones, and how variations in the structure and processing of Tg can lead to thyroid disease. Specific objectives for the next project period, and the experimental approach for each, are as follows: a. identify the donors of the outer iodothyronine rings, by sodium borohydride reduction, 14C incorporation, and peptide sequencing; b. locate the sites of iodine cleavage within the Tg polypeptide chain, and investigate their possible relation to hormonogenesis, by in vitro iodination of Tg, controlled enzyme digestion, peptide purification, and sequence identification; c. apply these and previous techniques to an examination of Tg's from human thyroid tissue available from surgery, particularly familial goiter; d. isolate and identify the thyroidal exopeptidases, and define their proteolytic roles, by enzyme purification and assay against Tg substrate; and e. examine Tg proteolysis in primary cultures of thyroid follicles. The proposed work should improve understanding of how thyroid hormones are synthesized and released, and of how changes in these processes relate to thyroid diseases, particularly familial goiter.